The goal of this research effort is to understand how important types of white blood cell, called T and B lymphocytes, recognize the presence of a microorganism or cancer cell in the body, or inappropriately recognizes a normal component of the body (an "auto-antigen"). This research tries to provide a detailed understanding of how the substances (antigens) making up these microorganisms, cancer cells, or normal self-components, are made visible to the defending lymphocytes cells or the auto-reactive cells. We have examined the structure and function of special cellular proteins called major histocompatibility complex (MHC) molecules that are essential for antigens to be recognized by T cells. Our previous work has described the events within a cell that bring together the antigen and MHC molecule and the cellular distribution of antigenic complexes within the body (antigen processing and presentation). By understanding these events, we will be able to identify the most important components of microorganisms or cancer cells to use in protective or therapeutic vaccines and how best deliver these substances to stimulate an effective immune response. We have made several advances in the past year. We previously reported a new method for the direct confocal microscopic visualization in real time of the interactions of T cells and antigen presenting cells in intact lymphoid tissue, showing that individual T cells bind stably to antigen-bearing dendritic cells for as long as 15 hrs. before activation-associated events lead to dissociation of the cell pairs and rapid migration of the T cells in the lymphoid tissue. Immunological synapses between T cells and a critical antigen presenting cell (the dendritic cell) were visualized in vivo as was clonal T cell division. This experimental system has now been improved by moving to multiphoton rather than confocal imaging, and initial success at true intravital observation has been achieved. Tumor metastasis models have been established for imaging anti-tumor effector function and preliminary results have been obtained imaging the bowel, which will enable direct investigation of the interaction of microorganisms with gut epithelium and the mucosal immune system. Ongoing studies are examining the movements of B cells in lymphoid tissues and the possible role of dendritic cells in presentation of antigen not only to T cells but also to B cells. Using high efficient methods for retroviral transduction of dendritic cells grown ex vivo, we have been able to modify the function of these key antigen presenting cells and to show that a particular signaling molecule called Traf6 plays a unique role in CD40-dependent signaling for secretion of the cytokine IL-12 that has a central role in inflammatory-type (Th1) T-cell responses. Finally, we have been able to construct and express a series of fluorescent protein chimeras in dendritic cells that enable us to viualize the changing distribution of key molecules involved in T cell antigen recognition, cell-cell adhesion, and costimulation during the interactions of T cells with dendritic cells.